Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Kramer, Michaela Andrea; Wetzel, Svava K.; Plückthun, Andreas; Mittl, Peer R. E.; Grütter, Markus G.

doi:10.1016/j.jmb.2010.09.023

Cited by 80 publications

(90 citation statements)

References 31 publications

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“…This observation is supported by results from our previous study of proteins with Ankyrin repeats, in which we observed a similar influence of the stability of capping repeats on the overall protein stability. 22,25,26 In fact, the Ising model predicts that the stability of these proteins arises from mutual stabilization of neighboring repeats, [25][26][27][28] and these effects are therefore expected to propagate throughout the entire protein.…”

Section: Discussionmentioning

confidence: 99%

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

et al. 2012

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A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature-and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5°higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

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Section: Discussionmentioning

confidence: 99%

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

et al. 2012

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“…The structure was determined by molecular and <B C-cap > = 42.30 Å 2 ), as previously observed for consensus-design ArmRPs [10,23] and other α-solenoid proteins [24][25][26] (Table 3). The largest temperature factors were found in the C-terminal capping repeat, especially within the loop connecting helices 2 and 3, that also displays no crystal contacts.…”

Section: Structure Of Computationally Designed Armrp Car2mentioning

confidence: 99%

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Reichen

Hansen

Forzani

et al. 2016

Journal of Molecular Biology

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Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library.Stacked armadillo repeats, each consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed curvature variations in natural ArmRPs, and identified a repeat pair from yeast importin-α as having the optimal curvature geometry to be complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry.Computationally designed armadillo repeat proteins (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics (MD) simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

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“…Selected DARPins from phage display and ribosome display were cloned into a pQE30-derived vector (Qiagen), containing an N-terminal MRGS(H) 6 tag and a C-terminal double stop codon (pQE30ss), or into pQE30_sfGFP, the latter to create DARPins with a C-terminal fusion of sfGFP. 22 For ELISAs, affinity measurements, labeling and flow cytometry, DARPins were produced in soluble form in E. coli XL1-blue (Stratagene, La Jolla, USA) and purified using Ni-NTA Superflow (Qiagen) in gravity-flow columns as described.…”

Section: Purification Of Darpinsmentioning

confidence: 99%

“…Recently, the C-terminal capping repeat was redesigned to further improve the stability of these proteins. 5,6 Their stable and yet adaptable structure, including the possibility to create hetero-oligomers of various geometries, small molecular weight and ease of production make them suitable tools for biomedical applications. Moreover, the introduction of unique cysteines allows site-specific modification and conjugation with various effector moieties.…”

Section: Introductionmentioning

confidence: 99%

DARPins Recognizing the Tumor-Associated Antigen EpCAM Selected by Phage and Ribosome Display and Engineered for Multivalency

Stefan

Martin-Killias

Wyss-Stoeckle

et al. 2011

Journal of Molecular Biology

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Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Cited by 80 publications

References 31 publications

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

DARPins Recognizing the Tumor-Associated Antigen EpCAM Selected by Phage and Ribosome Display and Engineered for Multivalency

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